JP2020517968A - Controlling molecules migrating through nanopores - Google Patents

Abstract

Abstract: To reduce unwanted variability in the rate of DNA migrating through solid-state nanopores, we used ultrathin nanoporous silicon nitride (NPN) membranes separated from one sensing nanopore by a nanoscale cavity. Show that the moving molecules are localized in advance at the nanoscale. Comprehensive experimental results show that the presence of this nanofilter minimizes the overall coefficient of variation of transit time in the sensing pore over a range of DNA sizes depending on cavity height. ing. With such sophisticated nanopore devices, the standard deviation of the transit time distribution is minimized, regardless of their diameter and stability. These results are also the first experimental validation showing that inter- and intra-molecular transit time variability depends on the conformational entropy of such molecules before migration, and solid state nanopores Provide a practical strategy for controlling transit through,

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority of US Provisional Application No. 62/491,572, filed April 28, 2017, the entire disclosure of which is hereby incorporated by reference. Incorporate.

The present disclosure relates to a nanopore device and a manufacturing method thereof.

When a biopolymer such as DNA alone moves through the nanopore, the dynamics of molecular transport becomes complicated. It is considered that the velocity at the time of passage depends not only on the conformation and proportion of the molecule outside the pores but also on the transient interaction with the pore wall and the membrane material and the influence of thermal fluctuation. There is. The end result is that the movement of molecules is affected by the breadth of the distribution of passage velocity due to both intra- and intra-molecular velocity fluctuations. This widening of the transit time complicates the simple conversion to find the position of a molecule from time and complicates the application to mapping. Will be significantly limited compared to.

The high level of motor control of stepwise movement of DNA in the pores using enzymes has been somewhat successful, and many years of intensive research now yield results in the form of biological nanopore-based sequencing devices. However, significant challenges remain in harnessing the potential offered by solid-state nanopores.

In experiments involving migration rate control, most efforts have involved a variety of means, including laser modulation of surface charge density to control electroosmotic flow, selection of appropriate electrolytes for both aqueous and ionic liquids, and viscosity. Focus on slowing down DNA by tailoring cells, contacting the pores with gels or using different membrane materials. These methods can slow the rate of DNA migration to varying degrees, but at the expense of broadening the transit time distribution.

A few studies have examined the factors that widen the transit time distribution. It has been found that the choice of salt solution has a great influence on the width of the transit time distribution. Simulation studies have demonstrated that as the electric field gradient widens, the equilibrium state of the polymer collapses, resulting in a wider transit time distribution than the equilibrium prediction results. Molecules that have become long before moving will have a longer transit time due to increased drag, and will accelerate as they approach the end of movement.

Unfortunately, the major mechanism responsible for the large variability in transit time is thought to be the large conformational entropy available in DNA molecules prior to translocation of the nanopore, but the geometry near the nanopore is sufficient. Experimental validation remains difficult due to the complexity of manufacturing devices that can be limited to

This section provides background information regarding the present disclosure that is not necessarily prior art.

This section presents a general summary of the disclosure and is not an exhaustive disclosure of its full scope or all features.

Systems are provided for controlling the movement of target molecules through the nanopore. The system includes a sensing structure and two chambers that are configured to contain a fluid and that are fluidly connected to each other by a fluid channel, the sensing structure being inserted into the fluid channel. As a result, the two chambers that are connected so that the fluid cannot pass between the two chambers other than passing through the nanopores formed in the sensing structure, are electrically connected to the voltage source, and are connected to the sensing structure. Two electrodes, one for each of the two chambers, configured to apply an electric potential are included.

A detection film is provided on the base material, and one nanopore is formed on the detection film. The sensing film may be composed of a dielectric material or a two-dimensional material. One or more spacers are disposed on the exposed upper surface of the sensing film. A filter film is disposed on the upper surface of the sensing film overlying the one or more spacers, whereby the sensing film, the one or more spacers and the filter film form a sensing structure. A plurality of nanopores are formed on the filter film.

In some embodiments, the filter membrane is separated from the sensing membrane by about the same distance as the contour length of the target molecule. For example, the one or more spacers are configured to separate the filter membrane from the sensing membrane by a distance that is sized to be less than the contour length of the target molecule.

In some embodiments, the average size of the nanopores in the plurality of nanopores formed in the filter membrane is less than twice the radius of gyration of the target molecule.

In some embodiments, the filter membrane is configured so that its electrical resistance is lower than that of the sensing membrane.

In some embodiments, there is a space between the filter membrane and the sensing membrane such that when the target molecule is coiled in free solution, the volume is less than 1000 times the volume of the target molecule. To do.

In some embodiments, the plurality of nanopores has a value of twice the distance between the filter membrane and the sensing membrane plus the average nearest neighbor distance between any two nanopores greater than the contour length of the target molecule. Is formed in the filter membrane at the average nearest neighbor distance between any two such nanopores.

In some embodiments, the plurality of nanopores is formed in a filter membrane such that the average nearest neighbor distance between any two nanopores is less than the radius of gyration of the target molecule when the target molecule is in free solution. Has been done.

In some embodiments, the filter membrane is configured to exhibit electrical resistance and the sensing membrane has an electrical resistance such that the quotient of the electrical resistance of the filter membrane divided by the electrical resistance of the sensing membrane is less than 0.01. It is configured as shown.

In one aspect, the sensing structure can be used to control the migration of the target polymer through the sensing membrane where one nanopore is formed. This method places the sensing structure in a fluid channel and drives the target polymer through the sensing structure's nanopores by applying an electrical potential to the sensing structure to determine the transit time of the target polymer through the sensing membrane's nanopores. The distance separating the sensing membrane and the filter membrane, including measuring, is shorter than the contour length of the target molecule.

In another aspect, the method positions a sensing structure within a fluid channel and drives a target polymer through the sensing structure's nanopores by applying an electrical potential to the sensing structure to drive the target through the sensing membrane's nanopores. Including measuring the transit time of the polymer, wherein the plurality of nanopores has an average nearest neighbor distance between any two nanopores, an average nearest neighbor distance between any two nanopores, and the target molecule is in free solution. It is formed on the filter membrane so that it is less than the radius of gyration of the target molecule.

In yet another aspect, the method drives a target polymer through the sensing nanopores of the sensing structure by applying an electrical potential to the sensing structure to enter a cavity formed between the filter membrane and the sensing membrane. , Trapping the target polymer in this cavity and evacuating the target polymer from the cavity of the sensing structure by reversing the potential applied to the sensing structure, the distance separating the filter membrane and the sensing membrane being the capture of the target polymer. Greater than radius.

Further areas of applicability will be apparent from the description provided herein. This summary description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.

The drawings described herein are for purposes of illustration of selected embodiments only, and are not intended to limit the scope of the present disclosure, but not all possible implementations.
FIG. 1 is a side view of an improved sensing structure for use in a nanopore translocation system. FIG. 2 is a top view showing a spacer having holes arranged in the detection film. FIG. 3A is a side view showing the movement of the filter film onto the detection film, and a gap is maintained between them by a spacer. FIG. 3B is a side view showing the movement of the filter film onto the detection film, and a gap is maintained between them by a spacer. FIG. 3C is a side view showing the movement of the filter film onto the detection film, and a gap is maintained between them by a spacer. FIG. 4 is a diagram of a setup example of the nanopore device. FIG. 5A is an exploded view of a support structure for a nanopore device. FIG. 5B is an exploded view of a support structure for a nanopore device. FIG. 6 is a side cross-sectional view of the support structure showing an enlarged view of the internal detection structure. FIG. 7A shows polymers of different lengths passing through the sensing structure. FIG. 7B shows polymers of different lengths passing through the sensing structure. FIG. 7C shows polymers of different lengths passing through the sensing structure. FIG. 7D is a corresponding graph showing the transit time distribution of each polymer. FIG. 7E is a corresponding graph showing the transit time distribution of each polymer. FIG. 7F is a corresponding graph showing the transit time distribution of each polymer. FIG. 8 is a graph showing that a polymer that is very long with respect to the dimensions of the nanodevice clogs the device by penetrating the sensing pores across more than one pore of the filter membrane. FIG. 9A is a graph showing the average transit time of the proposed nanodevice. FIG. 9B is a graph showing the average transit time of the control device. FIG. 10A is a graph showing the standard deviation of transit times for the proposed nanodevices. FIG. 10B is a graph showing the standard deviation of transit times for the control device. FIG. 11A is a graph showing the coefficient of variation of transit time of the proposed nanodevice. FIG. 11B is a graph showing the coefficient of variation of transit time for the control device. FIG. 12A is a graph showing a 500 bp DNA ladder within the pores of the proposed nanodevice. FIG. 12B is a graph showing a 500 bp DNA ladder within the pores of the proposed nanodevice. FIG. 12C is a graph showing a 500 bp DNA ladder within the pores of the control device. FIG. 13 is a graph showing the fold rates of various nanodevices associated with control devices. FIG. 14A is a graph showing proof-of-principle data for the operation of the proposed nanodevice as an entropy trap. FIG. 14B is a graph showing proof-of-principle data for the operation of the proposed nanodevice as an entropy trap.

Corresponding reference characters indicate corresponding parts throughout the several views of the drawings.

Exemplary embodiments will now be described more fully with reference to the accompanying drawings.

FIG. 1 shows an improved sensing structure 10 for use in a nanopore translocation system. The detection structure 10 is mainly configured by disposing the filter film 12 on the exposed upper surface of the detection film 14. It is preferable to dispose one or more spacers 13 on the upper surface of the detection film 14 and keep the filter film 12 away from the detection film 14 by the spacers 13. The distance separating the filter membrane from the sensing membrane is preferably about the same as the contour length of the target molecule (eg, monomer or polymer). The one or more spacers 13 serve to form a cavity between the filter membrane 12 and the sensing membrane 14. The cavity is typically a cavity whose volume is less than 1000 times the volume of the target molecule when coiled in free solution. A membrane is provided on the substrate 15 for structural support.

In the exemplary embodiment, sensing film 14 is preferably constructed of a dielectric material such as silicon nitride. Low pressure chemical vapor deposition (LPCVD) can be used to deposit a thin film of silicon nitride (eg, 5-100 nm, etc.) onto a substrate (eg, silicon wafer, etc.). The sensing film 14 may be composed of a two-dimensional material such as graphene or a transition metal dichalcogenide or other dielectric or non-dielectric material including a multi-layer metal/dielectric structure.

The spacer 13 is formed on the exposed upper surface of the detection film 14. In the exemplary embodiment, spacers 13 are formed by depositing a layer of silicon dioxide (eg, 50-1000 nm) on silicon nitride using plasma enhanced CVD (PECVD) or other deposition techniques. It Thereafter, the silicon dioxide layer is patterned with photoresist and selectively etched (eg, using dry RIE or wet HF) to help keep the filter film 12 and the sensing film 14 apart. One or more holes (ie, grids) are formed in the spacer 13 on 14. In the exemplary embodiment, the spacer 13 is a film in which a plurality of holes 11 are arranged, as best seen in FIG. In another embodiment, the spacer may be composed of a plurality of pillars provided side by side on the upper surface of the sensing film. Spacers and other arrangements for their manufacture are also contemplated by this disclosure.

A recess 17 is also provided on the opposite side of the substrate to expose the sensing film. Similarly, the photoresist 17 may be patterned on the base material and then etched to form the recesses 17 in the base material 15. In the exemplary embodiment, recess 17 is aligned with the center of spacer 13 located on the opposite side of the wafer. The recess 17 may be provided before or after the spacer 13 is formed, but it is easily understood that it is preferable that the recess 17 is provided before the filter film 12 is moved to the detection film 14.

The filter film 12 is also made of a dielectric material such as silicon nitride. In the exemplary embodiment, filter membrane 12 is a thin film (eg, on the order of 1 to 100 nanometers) that may be supported on substrate 15. The filter membrane 12 includes a plurality of nanopores 19 (also referred to as nanofilter pores herein) whose pore size can be adjusted in the range of 1 to 100 nanometers. The nanofilter pores preferably have an average size less than twice the radius of gyration of the target molecule. In one embodiment, the exemplary filter membrane 12 is manufactured by SiMPore, Inc. of West Henrietta, NY. Is commercially available from. The manufacture of such filter membranes is further described in the PCT patent application of PCT/US2014/051316, which is incorporated herein in its entirety. In other embodiments, the filter membrane 12 is manufactured using vapor phase sequential osmosis synthesis or other atomic layer deposition methods. Other methods of making filter membranes are also contemplated by this disclosure.

The filter film 12 can be deposited on the detection film 14 by covering the spacer 13 by bringing the filter film 12 into close contact with the detection film 14. In one example, van der Waals forces can be used to move the filter membrane 12 onto the sensing membrane 14. Referring to FIGS. 3A and 3B, the membranes are brought into close proximity (eg, a few microns), as seen in FIG. 3A. In some cases, the membranes may be in contact with each other. Each element may be cleaned (eg, by corona treatment, etc.) to render the surface hydrophilic.

In FIG. 3B, a stream of cold water vapor 31 is directed at the filter membrane 12 and into the cavity between the two membranes. The water between the membranes evaporates spontaneously. When the two membranes are pulled due to surface tension and the membranes come into contact, the membranes stick to each other. Finally, the substrate supporting the filter membrane 12 is mechanically removed. As can be seen in FIG. 3C, the filter membrane 12 has been peeled from the substrate 15 and remains on the spacer 13. To complete the detection structure, for example, the filter film 12 may be fixed to the detection film 14 by applying polydimethylsiloxane along the edge of the filter film 12 and bringing the filter film 12 into close contact with a suitable position. The freshly prepared sensing structure 10 may be cured by baking at 80 degrees Celsius.

Alternatively, a flow of compressed gas (eg, nitrogen or compressed air, etc.) directed at the filter membrane 12 can be utilized to move the filter membrane 12. This gas deflects the filter membrane 12 towards the spacers 13, creating contact once the membranes are in contact, such that the two membranes stick together (eg, by Van der Waals forces). It is possible to activate the respective surfaces with an air or oxygen plasma before the contact in order to promote the bonding of the two materials. Also, instead of applying polydimethylsiloxane or some other adhesive, the sensing structure 10 is thermally annealed at a high temperature while being compressed with a jig to thermally bond the filter film 12 to the spacers 13, thereby The need to strip the substrate from can be eliminated. Although a particular transfer method is mentioned above, other methods of depositing a filter film on the sensing film are also contemplated by the present disclosure.

In some embodiments, the sensing membrane 14 includes sensing pores 18 formed during fabrication of the sensing structure 10. For example, the sensing pores can be formed by electron beam drilling or ion beam drilling after depositing silicon nitride on the substrate. In other embodiments, the sensing pores 18 of the sensing membrane 14 may be formed using a controlled breakdown process in the context of nanodevices, as described further below. Other techniques for forming the sensing pores are also envisioned. Similarly, it is readily appreciated that other manufacturing methods can be used to form the sensing structure 10.

4-6 show an exemplary setup of the nanopore device 40. The nanodevice 40 is mainly composed of a support structure 41 in which various fluid passages are formed. In this example, the support structure 41 includes at least two chambers 42 that fluid can pass through by fluid channels 43. The two chambers 42 and the fluid channels 43 contain a fluid containing ions. In one example, the fluid is an aqueous potassium chloride solution. In another example, the fluid is a non-aqueous solvent such as a solution of lithium chloride in ethanol. It is easily understood that the composition of the fluid may vary depending on the analyte of interest.

The sensing structure 10 is inserted into the fluid channel 43. This prevents fluid from flowing between the two chambers except through the nanopores formed in the sensing structure 10. In this example, the support structure 41 is formed of two elements that are separately provided and mesh with each other. The sensing structure 10 fits into a recess formed in one or both of the two separate elements. The sensing structure 10 may be between two silicone gaskets 44. Other configurations of housing the sensing structure 10 in the support structure 41 and other configurations of the support structure 41 itself are also contemplated by the present disclosure.

Two electrodes 46 are inserted into the chamber 43 of the nanodevice one by one. These electrodes are electrically connected to a voltage source 49 and are configured to apply a potential to the sensing structure 10 located within the nanodevice 40. The voltage source 49 is controlled by the controller 48. In one embodiment, the controller is implemented by a data acquisition circuit electrically connected to a personal computer or another type of computing device. In some embodiments, the data acquisition circuit may be configured to measure the current flowing between the two chambers of the support structure. In other embodiments, the support structure 41 and/or the entire system can be placed in a grounded Faraday cage 47 to isolate it from electrical noise. Thus, this setup is similar to that commonly used for biomolecule detection in the nanopore sensing field.

As mentioned above, a controlled disruption process can be used to form the sensing pores in the sensing membrane. In this case, a potential is applied to the sensing membrane such that when the sensing structure 10 is within the support structure 41 of the nanopore device 40, a potential is induced in the sensing membrane that induces an electric field whose value exceeds 0.1 volts/nanometer. Is added. The controlled destruction of the material comprising the sensing film results in the formation of one nanopore in the sensing film. The leakage current in the sensing membrane is monitored while the potential is applied to the sensing membrane. The formation of nanopores is determined by the rapid increase in leakage current in the sensing membrane. When a sudden increase in leakage current is detected, the potential of the membrane disappears. Further details regarding the controlled destruction process can be found in US Patent Application Publication No. 2015/01080808, which is incorporated herein in its entirety.

Various applications are envisioned for the above nanodevices. For example, a mixture of analytes such as DNA or other charged molecules can be flowed through the sensing structure 10 from one chamber of the nanodevice 40 to the other chamber. For most applications, the analyte will enter from the filter membrane 12 side, but in some applications (ie, entropy cage) the analyte may enter from the sensing membrane 14 side. In operation, the analyte is driven to a small voltage (eg, up to 1 volt) to electrophoretically move through the system.

In an exemplary embodiment, the dimensions of the nanodevice and its sensing structure can be tailored to the application. The dimensions of nanodevices are defined as follows.
h: vertical distance between sensing membrane and nanofilter x: average nearest neighbor distance between any two pores of nanofilter L: contour length of polymer under study Y: diameter of hole in spacer material L<h Let the polymer that is a short polymer. The short polymer should completely enter the gap before being trapped in the sensing pores, with some loosening. Nanodevices still pass these polymers, but have little effect on their migration kinetics through the sensing pores.

A polymer satisfying h<L<3 (x/2+h) is a medium polymer. Medium polymers represent the range of lengths where nanodevices are most useful for sizing applications. The polymer here usually does not clog the sensing pores even though it penetrates the two nanofilter pores, but the migration kinetics are strongly influenced by the filter membrane.

A polymer that satisfies L>3 (x/2+h) is a long polymer. The long polymer penetrates the two nanofilter pores at both ends into the sensing pores and sticks to it when a constant electric field is applied, sooner or later clogging the sensing pores. Due to the small number of nanofilter pores actually used, the actual nearest neighbor pore distance x may differ from the average for a particular device. Therefore, the above polymer length ranges are in most cases only valid estimates. Depending on the device, the values may differ slightly at the boundary between medium and long polymers, but on average this works reasonably well. Y is constrained only in that it must be small enough to prevent the flexible nanofilter layer from contacting the sensing membrane, but by focusing the electric field lines better within the cavity, the polymer is It is also conceivable to reduce Y in order to achieve a larger electric field gradient that stretches.

In summary, when the polymer is of the medium length defined above, nanodevices work best to stretch the polymer without occluding. The nanodevice dimensions need to be adjusted so that the polymer of interest is in the medium range.

In one aspect of the present disclosure, the transit time distribution characteristics are controlled by appropriately configuring the nanodevice 40 according to a specific application. Short polymers are defined herein as polymers that are too short to penetrate two nanofilter pores 19 while still being trapped in the sensing pores. Referring to FIGS. 7A and 7D, for short polymers, the transit time distribution is approximately log-normal. As the polymer lengthens, a long transit time tail appears, as shown in FIGS. 7B and 7E. In FIGS. 7C and 7F, the longer polymer remains trapped in the sensing pores 18 and penetrates the two nanofilter pores 19. These longer polymers not only penetrate the two nanofilter pores, but also have a lengthwise flattening along the nanofilter membrane to increase the likelihood of friction and sticking as well as the transit time distribution. The long tail at can be significantly larger. For polymers that are long enough to penetrate more than one pore of a nanofilter and have both ends simultaneously in the sensing pore, the mode of clogging is predictable. In this mode, both ends of the polymer remain trapped in the two nanofilter pores and are trapped in the sensing pores, thereby clogging the sensor. This is an upper limit on the length of polymer that can be reliably studied in nanodevice 40 for a given spacer geometry.
A method for improving the migration of a target polymer on the sensing membrane of nanodevice 40 is described. As a starting point, the sensing structure is placed in the fluidic channel of the nanodevice as described above. Applying an electrical potential to the sensing structure drives the target polymer through the sensing structure nanopores. Next, the transit time of the target polymer passing through the nanopores of the sensing membrane is measured. Of note, one or more spacers are configured to separate the filter membrane from the sensing membrane and are sized such that the distance is less than the contour length of the target molecule. In some cases, the plurality of nanopores formed in the filter membrane are also configured so that the polymer is less likely to be clogged. For example, the dimensions of the filter membrane are designed as follows.

This is a sufficient boundary and assumes some looseness. A more robust boundary is 2h+x>L. That is, the plurality of nanopores in the filter membrane has an average nearest neighbor distance between any two nanopores that is twice the distance between the filter membrane and the sensing membrane plus an average nearest neighbor distance between any two nanopores. Is larger than the contour length of the target molecule.

For the range of polymers that can be effectively studied in the nanodevice 40, the filter membrane 12 can reduce the standard deviation of transit time and maximize the resolution of the transit time distribution as seen in FIGS. 9-11. This effect is caused by the elongation and elongation of the DNA as it passes through the filter membrane. Since the nanofilter pores are too small to pass through in the coil form, it is necessary to unwind and elongate the coil to pass through the filter. Once one end enters the space between the two membranes, an electric field gradient is created. This means that the tip of the polymer (closest to the sensing pores) experiences a higher tensile force than the rest of the polymer. This stretching mechanism also occurs without a filter membrane. However, coupled with the friction from contact with the filter membrane, the polymer is more stretched, reducing the number of conformations it can assume before entrapment. Since the polymer needs to stretch linearly to pass through the filter, the filter membrane limits the number of conformations that can be assumed during the passage of the polymer, resulting in highly consistent migration kinetics each time.

Although the control device still has an electric field gradient, it has a physical extension that is proportional to the square of the diameter of the sensing pores, which makes it more stretchable than the absence of the filter membrane, especially for smaller pores. Becomes incomplete. Thus, by using a filter membrane, there is no dependence on the size of the detection pores or the stability of the size, and even if all the pores are small pores, the analyte of interest can pass through. It becomes a very consistent sensor to detect. The filter membrane is essentially the mechanism by which the polymer is maximally stretched as the sensor approaches the sensing pores.

In an exemplary embodiment, the pores of the filter membrane are randomly distributed. Due to the fact that the nanofilter pores are randomly distributed, the length of the polymer that makes up the above segment may vary slightly from device to device depending on the local distribution of nanofilter pores near the sensing pores. There is. Nevertheless, it is possible to predict which length will be in which segment. In other embodiments, the pattern and position of the pores of the filter membrane can be controlled so that it can be predicted which length will be in which section.

In another example, clogging can be used to extend the time the polymer is in the sensing pores. For this application, it is necessary to tailor the dimensions of the nanodevice so that the polymer of interest has a long reach. A clogged polymer can be controllably moved by, for example, superimposing an AC field on a DC field while it is clogged. Prolonging the time the polymer is in the pores allows the use of lower bandwidth electronics for more accurate mapping of features along the length of the polymer and/or detection purposes.

Another application for the nanodevice 40 is nanopore size spectroscopy. The use of nanofilters minimizes the standard deviation of transit time (see Figures 9 and 10), which can be exploited to improve DNA size resolution for distinguishing populations mixed with DNA fragments. The important improvement here is that in the past, ordinary nanopores could only achieve a resolution of about 1000 bp in distinguishing DNA by length, and even with a strictly correct size of pores a resolution of about 400 bp (specifically, , 100 bp and 500 bp) can be achieved, but the proposed nanodevice 40 does not care about the size of the detection pores even if the detection pores are unstable with LiCl salt. The point is that a resolution of 500 bp can be realized in the range of a short polymer to a medium polymer length. Better performance (eg, about 100 bp) is expected when using different cationic species such as K ⁺ to shield the charge of DNA such as in KCl solution. Without a filter membrane, the distribution is highly dependent on the size of the sensing pores, which reduces device reliability and makes it more prone to failure, but having a filter membrane in place eliminates this dependency and reduces size. Increases the reliability of spectroscopy.

In another aspect of the disclosure, the proposed sensing structure can also be configured to inhibit migration of molecules in the folded state. Again, the sensing structure is placed in the fluidic channel of the nanodevice as described above. Applying an electrical potential to the sensing structure drives the target polymer through the sensing structure nanopores and measures the transit time of the target polymer through the sensing membrane nanopores. In this case, the average nearest neighbor distance between any two nanopores is constructed in a particular way to suppress folding. Specifically, a plurality of nanopores are formed in the filter membrane so that the average closest distance between any two nanopores is less than the radius of gyration of the target molecule when the target molecule is in the free liquid. There is.

Experimental data confirms an approach to suppress this folding. It is defined that the passage of a single file of DNA is a type 1 event, the event that DNA folds in the center and moves when it is completely folded is a type 2 event, and the event starts in the folded state The partially collapsed event that is defined is defined as a type 21 event. The folding rate is defined as the ratio of the time t ₂ spent in the folded state and the sum of the total transit time and the folded time t _total +t ₂ as follows.

This can be used in place of the partial location of the fold along the DNA backbone. F=0 for type 1 events and f=0.5 for type 2 events, with partially folded type 21 events in between. An example of each type can be found in the inset of FIG.
With continued reference to FIG. 13, two of the eight nanodevices studied were folded while the sensing pores were large enough to allow migration in the folded state. Note that it almost completely suppressed the movement in the closed state. However, the remaining six nanodevices behaved similarly to the control device (ie, no filter membrane). Since the suppression of folding occurred (or did not occur) at all DNA lengths studied in each device, it is supposed that the static property of the filter membrane is the factor that suppresses folding. Does not necessarily exist in all nanodevices. The most likely reason for this is that the pore sizes and positions of the nanofilters are randomly distributed.

Because DNA is a rigid polymer, it requires considerable force to fold into a nanopore. Due to the low impedance of the filter membrane, most of the voltage drops in the sensing pores and there is only a small voltage drop, and the force present in the filter membrane is small. Since the electric field decays with the square of the radial distance from the sensing pore as it moves away from the sensing pore, only nanofilter pores within a lateral distance range approximately equal to the height of the gap from the sensing pore, The voltage drop is sufficient to actually pass the DNA. For porosity considered, it means less than 8 nanofilter pores that actively allow DNA to pass through. If all nanofilter pores close to the sensing pore are very small (ie, defined as the persistence length of double-stranded DNA or less than about 30 nm), then the DNA will be passed through the filter in a folded configuration. Will not be strong enough. The time that the DNA is between the membranes is not enough to completely loosen, so if it were unfolded when it passed through the filter, the sensing pores would also pass unfolded. Cheap. In this case, the filter membrane is optimized by making the nanofilter pores small (<30 nm).

Alternatively, if the two nanofilter pores that are closest to the detection pore are so close that DNA molecules that pass through the nanofilter are almost always trapped in both pores at the same time from both ends, the The filter pores need to be linearized and selected (ie the situation as shown in Figure 7C). This process tends to linearize the DNA and facilitate capture in the unfolded state. This is because the polymer stretches against tensile forces before moving through the sensing pores. In this case, the filter membrane is optimized by having a very high porosity such that the average distance between the pores is shorter than the height of the gap between the membranes, and the nanofilter pores are not necessarily longer than the sustained length of the polymer. Even if it is not small, folding can be suppressed.

In this scenario, the polymer is very likely to penetrate the two nanofilter pores because h<<L because x<<L (eg, x<L/5) is required to suppress folding. Since /2 is required, the polymer is not long enough that both ends enter the sensing pore at the same time. Again, Y is only constrained in that it must be small enough to prevent the flexible filter membrane from contacting the sensing membrane. For example, x<68 nm and h>170 nm would be required to suppress the folding of 1000 bp dsDNA (340 nm). For the nanofilter properties used (thickness 50 nm), Y=1000 nm would work as well. As mentioned above, other dimensions can be limited.

Another way to control the migration of the target polymer is to enter and exit the cavity formed between the sensing membrane and the filter membrane of the sensing structure. By applying an electric potential to the sensing structure, the target polymer can be forced into the cavity. For example, when the nanodevice 40 is reversely operated so that DNA is captured from the detection pore side and drawn into the gap between the membranes, it functions as an entropy trap. Due to the small force exerted on the DNA in the gap between the nanofilter and the sensing membrane, the DNA is more likely to be trapped therein longer than if the nanofilter was not in place. Therefore, the target polymer is trapped in the cavity. In order to maximize this trapping time, it is important to make the gap h between the membranes at least as large as the capture radius of the detection pores (usually in the order of 100 to 1000 nanometers). This would cause the voltage drop across the nanofilter to be insufficient to eliminate the diffusion and the trap time would be very long. Although it is difficult to accurately estimate the capture radius, it is usually in the range of 100 to 1000 nm, so that it can be said that the requirements for dsDNA are h>1000 nm and d<30 nm. The target polymer is then ejected from the cavity by reversing the potential applied to the sensing structure. The difference with this device is that it can work with any length of polymer that fits in the gap.

Another tuning parameter to maximize the trapping time is to use very small nanofilter pores (eg <30 nm), which increases the entropy barrier to migration. The method of increasing porosity also reduces the electric field at the nanofilter, which makes escape more difficult. There is usually an interaction between nanofilter pore size and porosity, and these parameters need to be tuned together to maximize trapping time. The most important parameter for trap applications is the ratio of the electrical resistance of the active area of the nanofilter membrane to the electrical resistance of the sensing pores. For entropy trap applications, this value should be kept extremely small. For example, the ratio of the electrical resistance of the filter membrane to the electrical resistance of the sensing membrane is preferably less than 0.01.

FIG. 14 shows proof of principle data regarding the operation of these devices as entropy traps. This figure shows the loading of trapped molecules and their subsequent recovery. The nanocage may have applications as a nanoscale reactor. In this application, DNA is loaded into the trap through the sensing pores, where it is small enough to freely diffuse through the nanofilter into the space between the membranes and some of the DNA present on the nanofilter side of the system. Interact with small molecules of. The voltage can then be reversed to retrieve the reaction products and study.

Another possible application is to capture a low concentration sample in the intermembrane gap at high voltage to locally increase the concentration in the gap, then reverse and lower the voltage for more detailed studies. , There is a concentrator that recaptures at low voltage.

The filter membrane usually functions as a filter. These nanofilters allow very clear size exclusion for spherical molecules, while allowing linear polymers to pass even when the free solution size is larger than the nanofilter pores. It can be used as a filter to clean up real world samples (such as blood) containing a wide variety of biomolecules that clog the sensing pores. The size of the nanofilter pores can be adjusted to exclude all background molecules while passing only the target of interest. What is needed here is that the nanofilter pores are small enough to exclude the smallest particles (d<f) that need to be filtered and the target polymer studied is of short to medium length. In some respects, f is the diameter of the smallest particle that needs to be filtered. Therefore, the size of the nanopore filter depends on the application and the expected molecular size in the sample.

The fluid in the gap between the nanofilter and the sensing pores is necessarily stationary and there is no flow, so the nanofilter is used to contact the sensing pores even in the presence of cross flow above the nanofilter membrane. It is possible to create a shear-free region of the fluid to be used. Since no shear flow occurs at the location of the sensing pores, this allows for a high capture rate of the polymer by the sensing pores and on the nanofilter for the purpose of facilitating migration of new analytes near the pores. You can also use the flow in.

The above description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. The individual elements or features of a particular embodiment are usually not limited to that particular embodiment, but are interchangeable where applicable and selected embodiments without specific illustration or description. Can be used in. The same can be different in many ways. Such variations should not be considered as departures from the present disclosure, and all such modifications are intended to be included within the scope of the present disclosure.

The terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. As used herein, the singular may be intended to include the plural as well, unless the context clearly indicates otherwise. "Comprises," "comprising," "inclusion," "having" are inclusive, thus indicating the presence of a feature, an integer, a step, an operation, an element and/or a component, but one or more other It does not exclude the presence or addition of features, integers, steps, acts, elements, components and/or groups thereof. The steps, processes and acts of the methods described herein should not be construed as requiring performance in the particular order referred to or illustrated, unless otherwise specified as the order of performance. It should also be appreciated that additional or alternative steps may be used.

An element or layer is "above", "fits into," "connected with," "disposed over," or "coupled with" another element or layer. If directly on another element or layer, fitted on another element or layer, connected to another element or layer, arranged on another element or layer, It may be linked to other elements or layers or there may be intervening elements or layers. In contrast, when an element or layer is "immediately above", "directly fitted in", "directly connected to" or "directly connected to" another element or layer, There may be no intervening elements or layers. Other words used to describe relationships between elements need to be interpreted similarly (eg, "between" and "directly between", "adjacent" and "directly adjacent", etc.). As used herein, the term "and/or" includes any and all combinations of one or more of the items listed therein.

In this specification, the various terms such as first, second, third, etc. may be used to describe various elements, components, regions, layers and/or sections. , Layers and/or sections should not be limited to these terms. These terms are only used to distinguish one element, component, region, layer or section from another region, layer or section. As used herein, terms such as "first", "second", and other numerical terms do not imply order or order, unless context clearly indicates otherwise. Accordingly, a first element, component, region, layer or section described below is referred to as a second element, component, region, layer or section without departing from the teachings of the exemplary embodiments. You can

Spatial relative terms such as "inside", "outside", "underneath", "below", "below", "above", "above" are used herein for ease of explanation. Thus, it may be used to describe the relationship between one element or feature as shown and another element or feature. Spatial relative terminology may be intended to encompass other orientations of the device during use or operation in addition to the orientation shown. For example, when the illustrated device is flipped over, elements that are "below" or "just below" other elements or features are placed "above" other elements or features. Thus, examples of the term "bottom" include both top and bottom orientations. The device may be otherwise oriented (90 degrees or otherwise) and the spatially relative descriptors used herein interpreted accordingly.

Claims (21)

A system for controlling the movement of a target molecule through a nanopore, comprising:
A sensing film provided on the substrate and having one nanopore therein;
One or more spacers disposed on the exposed upper surface of the sensing membrane;
A filter film disposed on the upper surface of the detection film so as to cover the one or more spacers;
Two chambers that are configured to contain a fluid and that are fluidly connected to each other by a fluid channel,
Two electrodes,
Equipped with
The sensing membrane, the one or more spacers, and the filter membrane constitute a sensing structure,
The filter membrane has a plurality of nanopores formed therein,
The sensing structure is inserted into a fluid channel to prevent the fluid from passing between the two chambers except through the nanopores formed in the sensing structure,
The two electrodes are electrically connected to a voltage source and are configured to apply a potential to the sensing structure, such that one of the two electrodes is disposed in each of the two chambers.
system. The system of claim 1, wherein the filter membrane is separated from the sensing membrane by a distance that is about the same as the contour length of the target molecule. The system of claim 1, wherein the average size of the nanopores in the plurality of nanopores formed in the filter membrane is less than twice the radius of gyration of the target molecule. The system of claim 1, wherein the filter membrane is configured to have an electrical resistance lower than that of the sensing membrane. A space exists between the filter membrane and the detection membrane such that the volume of the target molecule is less than 1000 times the volume of the target molecule when the target molecule is coiled in a free solution. The system according to Item 1. The system of claim 1, wherein the sensing film is constructed on a dielectric material. The system of claim 1, wherein the sensing membrane is constructed on a two-dimensional material. 2. The one or more spacers of claim 1, wherein the one or more spacers are configured to separate the filter membrane from the sensing membrane by a distance, the distance being less than the contour length of the target molecule. system. The plurality of nanopores has a value such that a value obtained by adding twice the distance between the filter membrane and the sensing membrane and the average nearest neighbor distance between any two nanopores is larger than the contour length of the target molecule. 9. The system of claim 8, formed in the filter membrane at the average nearest neighbor distance between any two nanopores. The plurality of nanopores are formed in the filter membrane such that the average closest distance between any two nanopores is less than the radius of gyration of the target molecule when the target molecule is in free solution. The system of claim 1. The filter membrane is configured to exhibit electrical resistance, and the sensing membrane exhibits electrical resistance such that the quotient of the electrical resistance of the filter membrane divided by the electrical resistance of the sensing film is less than 0.01. The system of claim 1, wherein the system is configured to: A sensing structure for controlling the movement of a target molecule through a nanopore, comprising:
A sensing film with one nanopore formed,
One or more spacers disposed on the surface of the sensing membrane;
A filter film disposed on the surface of the detection film so as to cover the one or more spacers,
A plurality of nanopores are formed on the filter membrane,
The sensing structure wherein the one or more spacers are sized such that the filter membrane is separated from the sensing membrane by a distance that is approximately the same as the contour length of the target molecule. The sensing structure according to claim 12, wherein the filter film is configured to have an electric resistance lower than that of the detection film. A space exists between the filter membrane and the detection membrane such that the volume of the target molecule is less than 1000 times the volume of the target molecule when the target molecule is coiled in a free solution. Item 12. The sensing structure according to item 12. The plurality of nanopores has a value such that a value obtained by adding twice the distance between the filter membrane and the sensing membrane and the average nearest neighbor distance between any two nanopores is larger than the contour length of the target molecule. The sensing structure according to claim 12, wherein the sensing structure is formed in the filter membrane at the average nearest distance between any two nanopores. The plurality of nanopores are formed in the filter membrane such that the average closest distance between any two nanopores is less than the radius of gyration of the target molecule when the target molecule is in free solution. The sensing structure according to claim 12. The filter film is configured to have an electrical resistance, and the sensing film has an electrical resistance such that the quotient of the electrical resistance of the filter film divided by the electrical resistance of the sensing film is less than 0.01. 13. The sensing structure of claim 12, configured as A method for controlling the migration of a target polymer through a sensing membrane formed with one nanopore, comprising:
A sensing structure disposed on the sensing membrane is disposed in the fluid channel in a state where the filter membrane on which a plurality of nanopores are formed is separated from the sensing membrane by one or more spacers,
Driving a target polymer through the nanopores of the sensing structure by applying an electrical potential to the sensing structure,
Measuring the transit time of the target polymer through the nanopores of the sensing membrane,
The method wherein the distance separating the sensing membrane and the filter membrane is shorter than the contour length of the target molecule. For the plurality of nanopores of the filter membrane, the value obtained by adding the average nearest neighbor distance between the two nanopores to twice the distance between the filter membrane and the detection membrane becomes larger than the contour length of the target molecule. 19. The method of claim 18, wherein the average nearest neighbor distance between two such nanopores is formed. A method for controlling the migration of a target polymer through a sensing membrane formed with one nanopore, comprising:
A sensing structure disposed on the sensing membrane is disposed in the fluid channel in a state where the filter membrane on which a plurality of nanopores are formed is separated from the sensing membrane by one or more spacers,
Driving a target polymer through the nanopores of the sensing structure by applying an electrical potential to the sensing structure,
Measuring the transit time of the target polymer through the nanopores of the sensing membrane,
The plurality of nanopores are formed in the filter membrane such that the average closest distance between any two nanopores is less than the radius of gyration of the target molecule when the target molecule is in free solution. Method. A method for controlling the migration of a target polymer to a sensing structure of a nanodevice, comprising a filter membrane having a plurality of nanopores and a sensing membrane having one sensing nanopore formed therein.
Driving a target polymer through the sensing nanopores of the sensing structure by applying an electrical potential to the sensing structure into a cavity formed between the filter membrane and the sensing membrane,
Trapping the target polymer in the cavity,
Ejecting the target polymer from the cavity of the sensing structure by reversing the potential applied to the sensing structure, the distance separating the filter membrane and the sensing membrane is greater than the capture radius of the target polymer. Great, way.